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Bisphosphonates Directly Regulate Cell Proliferation, Differentiation, and Gene Expression in Human Osteoblasts1

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Bisphosphonates are widely used clinically to treat bone diseases in which bone resorption is in excess. However, the mechanism of bisphos- phonate action on bone is not fully understood. Studies of direct action of bisphosphonates on bone have been
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  [CANCER RESEARCH 60, 6001–6007, November 1, 2000] Bisphosphonates Directly Regulate Cell Proliferation, Differentiation, and GeneExpression in Human Osteoblasts 1 Gregory G. Reinholz, Barbara Getz, Larry Pederson, Emily S. Sanders, Malayannan Subramaniam, James N. Ingle,and Thomas C. Spelsberg 2  Departments of Biochemistry and Molecular Biology [G. G. R., B. G., L. P., E. S. S., M. S., T. C. S.] and Oncology [J. N. I.], Mayo Clinic, Rochester, Minnesota 55905 ABSTRACT Bisphosphonates are widely used clinically to treat bone diseases inwhich bone resorption is in excess. However, the mechanism of bisphos-phonate action on bone is not fully understood. Studies of direct action of bisphosphonates on bone have been limited mainly to their effects onbone-resorbing osteoclast cells, with implications that some activity maybe mediated indirectly through paracrine factors produced by the bone-forming osteoblast cells. Little is known about the direct effects of bisphos-phonates on osteoblasts. In this report, the direct actions of severalbisphosphonates on cell proliferation, gene expression, and bone forma-tion by cultured human fetal osteoblasts were examined. Osteoblast cellproliferation was decreased, and cytodifferentiation was increased in adose-dependent manner in cultures treated with the bisphosphonate pam-idronate. In addition, pamidronate treatment increased total cellularprotein, alkaline phosphatase activity, and type I collagen secretion inosteoblasts. Consistent with the above-mentioned findings, the rate of bone formation was also increased in osteoblasts cultured with pamidr-onate. The actions of two other bisphosphonates, the weak-acting etid-ronate and the potent new analogue zoledronate, were also compared withthe action of pamidronate on proliferation of immortalized human fetalosteoblast (hFOB) cells and rate of bone formation. Pamidronate andzoledronate decreased hFOB cell proliferation with equal potency,whereas etidronate decreased proliferation only at much higher concen-trations. Studies comparing EDTA and etidronate indicate that etidronatemay act indirectly on the hFOB cells by reducing free divalent ionconcentrations, whereas pamidronate and zoledronate appear to act onthe hFOB cells by a direct action. Both pamidronate and zoledronateincrease hFOB cell bone formation, whereas no increase is observed withetidronate and EDTA. Taken together, these observations strongly suggestthat treatment with pamidronate or zoledronate enhances the differenti-ation and bone-forming activities of osteoblasts. INTRODUCTION The bisphosphonates are a family of pyrophosphate analogues inwhich the oxygen linking the phosphates has been replaced by carbon(1). These compounds have high affinity for hydroxyapatite crystals(2) and are potent inhibitors of bone resorption (3). The bisphospho-nates are widely used to treat bone diseases in which there is an excessof bone resorption (4). Several structurally related bisphosphonateshave been synthesized by changing the two lateral chains on thecarbon or by esterifying the phosphate groups (1). The resultinganalogues vary extensively in antiresorptive potency, with analoguessuch as etidronate being the weakest, pamidronate being more potent,and the new analogue, zoledronate, being the most potent (1).The bisphosphonate pamidronate [(3-amino-1-hydroxypropylidene)bisphosphonate] is currently used for the treatment of hypercalcemiaof malignancy, Paget’s disease, osteolytic bone metastasis of breastcancer, and osteolytic lesions of multiple myeloma. Pamidronate hasintermediate antiresorptive potency between the weak-acting etid-ronate and the most potent analogue, zoledronate (1, 5).Although the bisphosphonates are commonly used clinically to treatbone diseases, the mechanism of action of these compounds on boneis not completely understood. At the tissue level, treatment withbisphosphonates leads to an increase in bone mineral density that hasbeen attributed to decreased bone turnover (3, 5–12). This observeddecrease in bone turnover appears to be due to decreased frequencyand resorption depth of the bone remodeling units (7, 10, 12). Highdoses of bisphosphonates can lead to impaired mineralization (6, 13).However, at lower levels of bisphosphonates, mineralization is nor-mal, and net osteoblast function is unimpaired, leading to a positivebone balance (7, 10, 12, 14).At the cellular level, bisphosphonates have been shown to havedirect effects on osteoclasts. Bisphosphonates can reduce osteoclastnumbers by inhibiting the proliferation and recruitment of osteoclastprecursors (15–18) and inducing apoptosis in macrophages and ma-ture osteoclast cells (19–22). In addition, bisphosphonates can di-rectly inhibit the bone-resorbing activity of osteoclasts (23, 24). Themechanism by which bisphosphonates act directly on osteoclasts andosteoclast precursors has been reported to be due, at least in part, toinhibition of the mevalonate pathway (22, 25–30).In addition to the direct effects of bisphosphonates on osteoclasts,there is evidence that these compounds also act on the osteoclastsindirectly through the osteoblasts (31–33). Osteoblasts are key regu-latory cells in bone that regulate bone cell differentiation and func-tions. It is likely that this indirect effect is due to modulation of osteoblast secretion of soluble paracrine factors that influence oste-oclast activity (34–38). New studies also suggest that bisphospho-nates can influence osteoblast function as well (39–43). However, theobserved effects differ, depending on the bisphosphonate and themodel system used.Because the complete mechanism of action of bisphosphonates isnot understood, and their actions on the important bone-formingosteoblasts are confusing, the effects of several bisphosphonates(etidronate, pamidronate, and zoledronate) on the latter regulatorycells were examined. The proliferation, differentiation, and boneformation (as measured by mineralization of nodules) by condition-ally immortalized hFOB 3 cells were examined and compared (44). Inaddition, the effects of EDTA on hFOB cell proliferation and miner-alization were examined because some bisphosphonates are known tochelate divalent ions. MATERIALS AND METHODS Materials.  Pamidronate was produced by Novartis Pharma AG (Basal,Switzerland). Etidronate was produced by MGI Pharm Inc. (Minnetonka, MN).Zoledronate was provided by Novartis Pharma AG. DMEM:Ham’s F-12medium (1:1) and Alizarin Red S stain were purchased from Sigma (St. Louis,MO). Fetal bovine serum was purchased from Summit Biotechnology (FortCollins, CO). Received 5/1/00; accepted 9/1/00.The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked  advertisement   in accordance with18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by the Mazza Foundation (M. S. and T. C. S.), Mayo Foundation (G. G. R.and B. G.), Howard Wagner Cancer Research Fund, and NIH Training Grant HD07108-22 (to G. G. R.). 2 To whom requests for reprints should be addressed, at Department of Biochemistryand Molecular Biology, 1601A Guggenheim Building, Mayo Clinic, 200 First StreetS. W., Rochester, MN 55905. Phone: (507) 284-4924; Fax: (507) 284-2053; E-mail:spelsberg.thomas@mayo.edu.  3 The abbreviation used is: hFOB, human fetal osteoblast. 6001 Research. on November 12, 2015. © 2000 American Association for Cancercancerres.aacrjournals.org Downloaded from   Cell Culture.  The hFOB cells were developed previously and character-ized in this laboratory (44). Briefly, hFOB cells were derived from primarycultures of fetal tissue and conditionally immortalized with a gene coding forthe temperature-sensitive mutant (ts A58) of the SV40 large T-antigen. Thiscell line was isolated from primary cultures based on its osteoblast phenotype.Incubation of hFOB cells at the permissive temperature (34°C) results in rapidcell division, whereas little or no cell division occurs at the restrictive tem-perature (39°C). hFOB cells were maintained at 34°C in DMEM:Ham’s F-12medium (1:1) supplemented with 10% (v/v) fetal bovine serum and 300   g/mlGeneticin. Culture medium was removed and replaced with fresh mediumevery 3 or 4 days during experimentation. Cell Proliferation.  hFOB cells were seeded at 20,000 cells/cm 2 in 96-wellplates and incubated at 34°C for 24 h in normal culture medium. The mediumwas then replaced with 100   l/well fresh medium containing various concen-trations of pamidronate. The relative number of viable cells in each well wasthen determined at various times after treatment using the Cell Titer 96 AQ ueous One Solution Cell Proliferation Assay (Promega, Madison WI). Briefly, 20   lof Cell Titer 96 AQ ueous  One Solution were added to each well, including threewells containing only medium for background substraction. The cells werethen incubated at 37°C for 30 min. The absorbance at 490 nm in each well wasthen determined using a SpectraMax 340 plate reader/spectrophotometer (Mo-lecular Devices Corp., Sunnyvale, CA). This technique was determined toproduce a linear relationship between the number of viable hFOB cells and theabsorbance at 490 nm. Total Cellular Protein.  hFOB cells were seeded at 20,000 cells/cm 2 in12-well and 96-well plates and incubated at 34°C for 24 h in normal culturemedium. The medium was then replaced with fresh medium containing variousconcentrations of pamidronate. The cells in the 12-well plates were rinsedtwice with 1  PBS, and the total protein was determined in cell lysates usingthe Bio-Rad protein assay (Biorad Laboratories, Hercules, CA). The totalprotein values were normalized to the relative number of viable cells asdetermined directly in the 96-well plates using the above-mentioned prolifer-ation assay. Alkaline Phosphatase Activity.  hFOB cells were seeded at 20,000 cells/ cm 2 in 12-well and 96-well plates and incubated at 34°C for 24 h in normalculture medium. The medium was then replaced with fresh medium containingvarious concentrations of pamidronate. Alkaline phosphatase activity wasdetermined in the 12-well plates using the Alkaline Phosphatase Kit (Sigma).The alkaline phosphatase activity values were normalized to the relativenumber of viable cells as determined directly in the 96-well plates using theabove-mentioned proliferation assay. Type I Collagen Secretion.  hFOB cells were seeded at 20,000 cells/cm 2 in 12-well and 96-well plates and incubated at 34°C for 24 h in normalculture medium. The medium was then replaced with fresh medium con-taining various concentrations of pamidronate. The amount of collagen typeI COOH-terminal propeptide was determined in the conditioned mediausing the Prolagen-C assay (Metra Biosystems, Inc., Mountain View, CA).The type I collagen values were normalized to the relative number of viablecells as determined directly in the 96-well plates using the above-men-tioned proliferation assay. Mineralization.  hFOB cells were seeded at 20,000 cells/cm 2 in 12-welland 96-well plates and incubated at 34°C for 24 h in normal culture medium.The medium was then replaced with fresh medium containing various con-centrations of pamidronate. The degree of mineralization was determined inthe 12-well plates using Alizarin Red staining. Briefly, medium was aspiratedfrom the wells, and the cells were rinsed twice with PBS. The cells were fixedwith ice-cold 70% (v/v) ethanol for 1 h. The ethanol was removed, and thecells were rinsed twice with deionized water. The cells were then stained with40 m M  Alizarin Red S in deionized water (adjusted to pH 4.2) for 10 min atroom temperature. The Alizarin Red S solution was removed by aspiration, andthe cells were rinsed five times with deionized water. The water was removedby aspiration, and the cells were incubated in PBS for 15 min at roomtemperature on an orbital rotator. The PBS was removed, and the cells wererinsed once with fresh PBS. The cells were then destained for 15 min with 10%(w/v) cetylpyridinium chloride in 10 m M  sodium phosphate (pH 7.0). Theextracted stain was then transferred to a 96-well plate, and the absorbance at562 nm was measured using a SpectraMax 340 plate reader/spectrophotometer(Molecular Devices Corp.). The concentration of Alizarin Red S staining in thesamples was determined by comparing the absorbance values with thoseobtained from Alizarin Red S standards. The mineralization values werenormalized to the relative number of viable cells as determined directly in the96-well plates using the above-mentioned proliferation assay. Statistical Analysis.  Significance was determined using the two-tailedStudent’s  t   test. RESULTSEffect of Pamidronate on hFOB Cell Proliferation.  Dose-response and time-course experiments were performed to determinethe effects of pamidronate on hFOB cell proliferation. As shown inFig. 1  A , treatment of hFOB cells with pamidronate for 6 days de-creased the number of viable hFOB cells in the cultures in a dose-dependent manner compared with vehicle-treated cells. A maximum95% reduction in viable hFOB cells was observed at the 25   g/mldose level. The differences in viable cell numbers were significant atthe 0.5   g/ml dose level, with  P    0.05 and  P    1    10  9 at thehighest dose (100   g/ml). The effect of pamidronate (0, 2.5, 10, and25   g/ml) on the proliferation of hFOB cells in culture over time isillustrated in Fig. 1  B . Whereas the control osteoblast cells continue toproliferate rapidly through 10 days of culture, the proliferation of theosteoblasts cultured with 2.5 and 10   g/ml pamidronate is slowed. Atconcentrations of pamidronate greater than 10   g/ml, the cells be-come rounded and detached, and significant osteoblast cell death isobserved. Fig. 1. Effect of pamidronate on hFOB cell proliferation. hFOB cells were seeded in96-well plates and cultured at 34°C in normal growth medium with or without pami-dronate. hFOB cell proliferation was then assessed as described in “Materials andMethods.”  A , dose response to pamidronate analyzed at day 6 (  ,  P    0.05;   , P  1  10  6 ,   ,  P  1  10  9 ) compared with vehicle treatment).  B , time course of pamidronate treatment analyzed at days 0, 3, 6, and 10.  E , vehicle;  ■ , 2.5   g/ml;  ‚ , 10  g/ml; and F , 25   g/ml pamidronate (  ,  P  0.05;   ,  P  0.0001;   ,  P  1  10  6 compared to vehicle treatment). The data represent the mean values ( n  4).  Error bars ,SDs from the mean values. 6002 EFFECTS OF BISPHOSPHONATES ON OSTEOBLASTS Research. on November 12, 2015. © 2000 American Association for Cancercancerres.aacrjournals.org Downloaded from   Effect of Pamidronate on Total Protein Levels in hFOB Cells. Fig. 2  A  illustrates the effect of increasing concentrations of pamidr-onate on total protein in hFOB cells. As shown, the total cellularprotein levels in the cultured hFOB cells are increased with increasingconcentrations of pamidronate after treatment for 4 days. This changein total cellular protein levels becomes significant at 1.0   g/ml pam-idronate ( P  0.05), and a maximum increase of 82% is observed at10   g/ml pamidronate. As demonstrated in Fig. 2  B , the increase intotal cellular protein induced by pamidronate (10  g/ml) is significantby day 3 ( P    0.001), reaches a maximum on day 5, and begins todecrease on day 7. Effect of Pamidronate on Alkaline Phosphatase Activity inhFOB Cells.  Dose-response and time-course experiments were per-formed to examine the effects of pamidronate on alkaline phosphataseactivity in hFOB cells. As shown in Fig. 3  A , pamidronate treatmentfor 4 days increased the alkaline phosphatase activity per viable hFOBcell in a dose-dependent manner. The increase in alkaline phosphataseactivity was significant at 2.5   g/ml pamidronate ( P    0.002) andreached a maximum of 38% over vehicle-treated cells at 10.0   g/mlpamidronate ( P  1  10  5 ). Fig. 3  B  shows the effect of pamidronate(10   g/ml) treatment of hFOB cells on alkaline phosphatase activityover time. The increased alkaline phosphatase activity was observedby day 2 and reached a maximum of 82% on day 5. After day 5, thepamidronate-induced alkaline phosphatase activity begins to decrease. Effect of Pamidronate on Type I Collagen Secretion fromhFOB Cells.  Fig. 4  A  demonstrates the effect of pamidronate treat-ment for 4 days on type I collagen secretion from hFOB cells.Pamidronate treatment caused a dose-dependent increase in type Icollagen secretion per viable hFOB cell. The minimally effectiveconcentration was 2.5  g/ml pamidronate ( P  0.01), and a maximumincrease of 65% was observed at the highest concentration used, 10.0  g/ml pamidronate ( P    0.01). Fig. 4  B  demonstrates the effect of pamidronate (10   g/ml) treatment on type I collagen secretion fromhFOB cells over time. Although type I collagen secretion in pamid-ronate-treated cultures was initially less than control in the experimentshown, type I collagen secretion gradually increased over the 7-dayculture period to levels above those of control cultures. Effect of Pamidronate on hFOB Cell Mineralization.  To exam-ine the effects of pamidronate on bone formation, hFOB cells werestained for calcium incorporation using Alizarin Red. As shown inFig. 5  A , treatment with pamidronate for 7 days increased the amountof staining per viable cell in a dose-dependent manner. The maximumeffect on mineralization was achieved at 10   g/ml pamidronate. Atthis concentration of pamidronate, an increase of 81% in Alizarin Redstaining per viable cell was observed ( P    1    10  5 ). As demon-strated in Fig. 5  B , the increase in mineralization induced by pamid- Fig. 2. Effect of pamidronate on total cellular protein in hFOB cells. hFOB cells wereseeded in 12-well and 96-well plates and cultured at 34°C in normal growth medium withor without pamidronate. The total protein was determined in the 12-well plates andnormalized to the relative number of viable cells as determined directly in the 96-wellplates as described in “Materials and Methods.”  A , dose response of pamidronate treat-ment analyzed on day 3 (  ,  P  0.05 and   ,  P  0.001 compared with vehicle treatment).  B , time course of pamidronate (10   g/ml) treatment analyzed on days 1, 2, 3, 4, 5, 6,and 7 (  ,  P  0.05 and   ,  P  0.001 compared with vehicle treatment). The data shownrepresent the mean values ( n    4).  Error bars , SDs from the mean values.  ■ , datanormalized to relative cell number;   , data not normalized.Fig. 3. Effect of pamidronate on alkaline phosphatase activity in hFOB cells. hFOBcells were seeded in 12-well and 96-well plates and cultured at 34°C in normal growthmedium with or without pamidronate. Alkaline phosphatase activity was determined in the12-well plates and normalized to the relative number of viable cells as determined directlyin the 96-well plates as described in “Materials and Methods.”  A , dose response topamidronate analyzed on day 4 (  ,  P    0.002 and   ,  P    1    10  5 compared withvehicle).  B , time course of pamidronate (10   g/ml) treatment analyzed at days 1, 2, 3, 4,5, 6, and 7 (  ,  P    0.05;   ,  P    0.01; and   ,  P    0.001 compared with vehicle).The data shown represent the mean values ( n    3 or 4).  Error bars , SDs from themean values.  ■ , data normalized to relative cell number;   , data not normalized. 6003 EFFECTS OF BISPHOSPHONATES ON OSTEOBLASTS Research. on November 12, 2015. © 2000 American Association for Cancercancerres.aacrjournals.org Downloaded from   ronate (2.5   g/ml) occurs between day 4 and day 10 of culture andbegins to decrease by day 14. Comparative Effects of Etidronate, Pamidronate Zoledronate,and EDTA on hFOB Cell Proliferation.  Because the bisphospho-nates can form insoluble complexes with divalent ions, the effects of the bisphosphonates etidronate, pamidronate, and zoledronate onhFOB cells were compared with those of EDTA on an equivalentmolarity basis. As shown in Fig. 6  A , these compounds inhibit hFOBcell proliferation with different potencies. Pamidronate and zole-dronate were approximately equally potent with 50% effective doses(ED 50 ) of 4.2    10  5 and 4.0    10  5 M , respectively. This findingdoes not correlate with the reported 100-fold higher  in vivo  potencyreported for zoledronate compared with pamidronate (5). Etidronatewas approximately 180-fold less potent than zoledronate and pamid-ronate, with an ED 50  of 7.5    10  3 M . The potency differencebetween etidronate and pamidronate is very similar to the reported  invivo  antiresorptive potency difference for these compounds (1). Theantiproliferative potency of EDTA was between that of zoledronate/ pamidronate and etidronate, with an ED 50  of 5.6  10  4 M .Because EDTA was as effective at similar concentrations as theweak bisphosphonate etidronate, it is possible that some of the ob-served effects of these compounds in culture are due to a reduction inthe free concentration of divalent ions in the culture medium. If thisis the case, we hypothesized that the addition of divalent ions to thebisphosphonate-treated culture medium would reverse the effects of the bisphosphonates on the osteoblast cells. To test this hypothesis,hFOB cells were treated with vehicle, etidronate, pamidronate, zoled-ronate, or EDTA at the respective ED 50  concentrations describedabove. The culture media were then treated with increasing concen-trations of calcium and magnesium chlorides, and proliferation of thehFOB cells was measured after 7 days of culture. As shown in Fig. 6  B ,the addition of increasing concentrations of divalent ions to theetidronate- and EDTA-treated culture media caused a dose-dependentincrease in hFOB cell proliferation, suggesting that etidronate wasinhibiting the hFOB cell proliferation by chelating the essential diva-lent ions. Interestingly, similar to the vehicle-treated cultures, theaddition of increasing concentrations of divalent ions to the pamid-ronate- and zoledronate-treated cultures caused a further decrease inhFOB cell proliferation. We conclude from this experiment that theantiproliferative effects of both EDTA and etidronate on hFOB cellsare likely due to reductions in the free divalent ion concentrationsavailable in the culture medium caused by these compounds. How-ever, the antiproliferative effects of the more potent bisphosphonates, Fig. 4. Effect of pamidronate on type I collagen secretion from hFOB cells. hFOB cellswere seeded in 12-well and 96-well plates and cultured at 34°C in normal growth mediumwith or without pamidronate. The amount of type I COOH-terminal propeptide wasdetermined in the conditioned media from 12-well plates and normalized to the relativenumber of viable cells as determined directly in the 96-well plates as described in“Materials and Methods.”  A , dose response to pamidronate analyzed on day 4 (  ,  P  0.01compared to vehicle).  B , time course of pamidronate (10   g/ml) treatment analyzed ondays 1, 2, 3, 4, 5, 6, and 7 (  ,  P  0.05;   ,  P  0.005; and   ,  P  0.002 comparedwith vehicle treatment). The data shown represent the mean values ( n  4).  Error bars ,SDs from the mean values.  ■ , data normalized to relative cell number;   , data notnormalized.Fig. 5. Effect of pamidronate on hFOB cell mineralization. hFOB cells were seeded in12-well and 96-well plates and cultured in normal growth medium with or withoutpamidronate. The degree of mineralization was determined in the 12-well plates usingAlizarin Red staining and normalized to the relative number of viable cells as determineddirectly in the 96-well plates as described in “Materials and Methods.”  A , dose responseto pamidronate analyzed at day 7 (  ,  P  0.02;   ,  P  0.005;   ,  P  0.001; and   , P    1    10  5 compared with vehicle treatment).  B , time course of pamidronate (2.5  g/ml) treatment analyzed on days 4, 6, 8, 10, and 14 (  ,  P    0.005;   ,  P    0.0005;and   ,  P    1    10  5 compared to vehicle treatment). The data shown represent themean values ( n  3 or 4).  Error bars , SDs from the mean values. ■ , data normalized torelative cell number;   , data not normalized. 6004 EFFECTS OF BISPHOSPHONATES ON OSTEOBLASTS Research. on November 12, 2015. © 2000 American Association for Cancercancerres.aacrjournals.org Downloaded from   pamidronate and zoledronate, appear to be due to a mechanism otherthan reduction of free divalent ions in the culture medium.The effects of etidronate, pamidronate, zoledronate, and EDTA onthe rate of bone formation as measured by hFOB cell mineralizationof the osteoblast-produced matrix was also examined. As shown inFig. 6 C  , the addition of pamidronate or zoledronate to the culturemedium produced a marked increase the Alizarin Red stain per viablecell. Similar to the results in the proliferation experiments, bothpamidronate and zoledronate displayed similar potencies in increasingmineralization in hFOB cells. Neither EDTA nor etidronate waseffective in increasing the mineralization of hFOB cells, even at veryhigh concentrations. Thus, the more potent bisphosphonates, pamid-ronate and zoledronate, have marked effects on both hFOB cellproliferation and the rate of bone formation that do not appear to bedue to loss of free divalent ion concentrations in the culture mediumbut rather act on some other pathway in the hFOB cells. The effectsof these more potent bisphosphonates are in sharp contrast to theeffects of the less potent bisphosphonate etidronate, which inhibitshFOB cell proliferation only at very high concentrations (apparentlyby reducing free divalent ion concentrations in the medium) andshows no effect on hFOB cell mineralization. DISCUSSION This study describes the effects of several bisphosphonates on theproliferation and differentiation of cultured hFOB cells. The hFOBcells are unique among the osteoblast models currently used in thatthey are conditionally immortalized normal human osteoblasts (44).Karyotype analysis on these cells revealed that they have only minorchromosomal translocations and deletions, which is in sharp contrastto the major chromosomal abnormalities that we observed in MG-63osteosarcoma cells by comparison. 4 Thus, the hFOB cells make anexcellent model system to study the function of osteoblasts and theeffects of agents such as bisphosphonates on osteoblasts  in vitro .The actual concentration ranges of the bisphosphonates that osteo-blasts and other cells in the body are exposed to under pharmacolog-ical conditions are unknown. Therefore, it is difficult to design  in vitro experiments that can directly correlate to physiological conditions.The concentrations of bisphosphonates used in these experimentswere chosen based on reported levels in patient sera after i.v. admin-istration of pamidronate (45–49) that reached transient concentrationsranging up to 4.29  g/ml or 10  5 M  in sera. Whereas these peak serumlevels are transient, bisphosphonates accumulate rapidly and at highconcentrations in bone (1). One report has also estimated that phar-macological doses as of one bisphosphonate, alendronate, could giverise to local concentrations as high as 1 m M  (10  3 M ) alendronate inthe resorption space (50). Using concentrations similar to the reportedpeak serum concentrations, we have observed direct effects of pam-idronate on cultured hFOB cells.The proliferation of hFOB cells was decreased in a dose-dependentmanner in cultures treated with pamidronate. This observation is notsurprising because similar effects of bisphosphonates have been re-ported with many other cell types including osteoclasts (20–22),intestinal epithelial cells (51), lymphocytes (52), macrophages (16,19) myelomas (53, 54) breast cancer cells (55), and primary osteo-blasts (39, 41). Recent experiments in this laboratory confirm thisinhibition,  i.e. , pamidronate treatment decreased the proliferation of cultured MCF-7 and T47D breast cancer cells as well as LnCAPprostate cancer cells. 5 In contrast, bisphosphonates have been reportedto induce proliferation of marrow osteoprogenitors (40) and inhibitapoptosis of osteocytes and osteoblasts (43). The reason for theseopposing effects is unknown but may be due to differences in the cell 4 M. Subramaniam, S. Jalal, S. Harris, M. Bolander, and T. C. Spelsberg, Comparisonof chromosomal alterations in normal and transformed human osteoblasts using multicolorspectral karyotyping, manuscript in preparation. 5 G. G. Reinholz, B. Getz, E. S. Sanders, J. N. Ingle, and T. C. Spelsberg, unpublishedobservations.Fig. 6. Comparative effects of bisphosphonates and EDTA on hFOB cell proliferationand mineralization. hFOB cells were seeded in 12-well and 96-well plates and cultured at34°C for 7 days in normal growth medium containing various concentrations of bisphos-phonates or EDTA. hFOB cell proliferation and mineralization were assessed as describedin “Materials and Methods.”  A , comparative effects of bisphosphonates and EDTA onhFOB cell proliferation.  } , EDTA;  ■ , etidronate;  ‚ , pamidronate;  F , zoledronate.  B ,effect of divalent ions on the antiproliferative effects of bisphosphonates and EDTA.hFOB cells were treated with etidronate (7.5    10  3 M ), pamidronate (4.2    10  5 M ),zoledronate (4.0    10  5 M ), and EDTA (5.6    10  4 M ) plus various concentrations of calcium chloride and magnesium chloride.  C  , comparative effects of bisphosphonates andEDTA on hFOB cell mineralization.  } , EDTA;  ■ , etidronate;  ‚ , pamidronate;  F ,zoledronate. The data shown represent the mean values ( n  4). 6005 EFFECTS OF BISPHOSPHONATES ON OSTEOBLASTS Research. on November 12, 2015. © 2000 American Association for Cancercancerres.aacrjournals.org Downloaded from 
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